Neutrophilic airway inflammation is associated with increased asthma severity. Nearly 50% of patients with asthma are non-eosinophilic and thus less likely to respond completely to corticosteroid therapy or current biologics, illustrating the unmet need to improve our understanding of this group of patients. Despite these associations, the pathways involved in neutrophilic asthma (NA) are only partially understood. MicroRNAs (miRNAs) exert a powerful effect on gene regulation and have been implicated in T helper 2 airway inflammation in asthma. However, there is a knowledge gap in our understanding of the role of miRNAs in NA. MiR-223-3p is associated with severe NA and leads to airway epithelial changes, implicating miRNAs in NA. Work by our group at the Yale Center for Asthma and Airway Disease cohort (YCAAD) found that miR-223-3p belongs to a network of a dozen miRNAs in sputum cells associated with airflow obstruction, asthma hospitalizations, decreased asthma quality of life, lymphocyte and neutrophil counts in the sputum. To understand this cell-specific association we performed in situ hybridization to identify sputum cells expressing miR-223-3p and found that neutrophils express high levels of miR-223-3p. This miRNA is positively correlated with toll-like receptor pathways and IL-17 expression in patients with asthma. We hypothesize that the sputum miRNA network is involved in the regulation of the Th17 pathway and neutrophilic airway inflammation in asthma. We will utilize two well characterized cohorts, YCAAD and the New York University/Bellevue Asthma Registry (NYUBAR), to execute the following aims: Aim 1. To determine the longitudinal expression of the sputum miRNA network and its role in neutrophilic airway inflammation. This aim will determine the stability of miRNA expression in the YCAAD cohort and will validate the expression of the miRNA network in the NYUBAR cohort. Aim 2. To define the network miRNAs released in extracellular vesicles and their association with neutrophilic airway inflammation. This aim will determine the extent to which unique network miRNAs are released into the extracellular environment via extracellular vesicles (EVs) and exert their functional role in the regulation of Th17 inflammation in airway epithelial cells. Aim 3. To determine the effect of the miRNA network in airway epithelial gene expression and its contribution to neutrophilic airway inflammation. This aim will elucidate how the miRNAs in the network interact with each other and how their regulatory role converges on specific inflammatory pathways. These studies will investigate a poorly understood asthma endotype using a novel paradigm of miRNA regulation of neutrophilic airway inflammation. The results derived from this project will lay the foundation for the improved identification of gene regulatory networks involved in NA and lead to potential therapeutic manipulation of miRNAs in neutrophilic airway inflammation.